In previous years we have proposed a model of four core domains of cytochrome c (cyt.c) fragment complexes whose interactions drive folding. A core domain is structural region containing a hydrophobic core and the surrounding shell, it folds and unfolds as a unit. To gain insight into this hypothetical core domain-domain interaction we have carried out site directed mutagenesis of yeast iso-2cyt. c: single substitutions L91, 120V, M64L, L85I, and M98L, double L91/M64L, 120V/M98L, and M64L/M98L, triple L91/120V/M98L L91/M64L/M98L, and 120V/M64l/M098L, quadruple L91/120V/M64L/M98L, and quintuple L91/120/M64L/L85I/M98L. These residues are located in the core or partly in athe core. Shell residue substitutions T99K were also performed. All these substitutions are those which occur going from yeast iso-2 to horse cyt.c. Despite these 'native' substitutions, single mutant 120V nd quadruple mutant L9I/120V/M64L/M98L iso-2 cyts. c are found to be non-functional (yeast cells containing phagemids bearing and the mutant gene fail to grow on non-fermentable carbon source, glycerol), while all the other mutants iso-2 cyts.c are functional. Thermal transition of the 695 nm absorption band of the wild type and mutant cysts. c, indicative of the Met 80S-heme Fe bond, show that there are interactions between Ile 20 and met 98 and between Met 64 and Met 98. The pattern of this transition and the data of the 628 nm absorbance band of the mutant cyts. c also suggest that residues 9 and 64 may influence each others substitution effect. Examination of the 655 nm absorption band of ht wild type and mutant cyts. c suggests that an increase in the intensity of this band partially correlates with the loss of function. All these observations can be explained by assuming that there may be some long-range interaction- network associated with the packed or ordered core residues of yeast iso- 2-cyt. c that modulates the stability of the conformation and the function. It is also assumed that these interactions are sensitive to substitution of the residues involved. Consistently, the collaborative two-dimensional NMR studies of the wild type and M64L, M98L and M64L/M98L mutant iso-2 cyts. c suggest that perturbation of the core residue packing by these substitutions, particularly by the double substitution significantly increases conformational mobility.